The recent discovery of HIV coreceptors has provided a new and dynamic vision of HIV envelope protein function. We have expanded on this concept to propose that the transient envelope protein interactions and conformational changes that arise during CD4- and coreceptor-mediated binding and fusion may expose critical targets for neutralizing antibodies, especially antibodies capable of neutralizing primary isolates (PI) of HIV. In this regard, we have developed novel 'fusion-competent' vaccines that elicit potent PI virus neutralizing antibodies when used to immunize transgenic mice that are tolerant to the human CD4 and CCR5 components of the vaccine. The finding of PI virus neutralization is in marked contract our results using comparable vaccines that present non-functioning envelope protein. In this proposal, we will firstly define the requirements and limits of PI virus neutralization by 'fusion-competent' vaccines. Secondly, we will extend these studies to identify the molecular structures and conformations that mediate HIV binding and fusion, and that serve as targets for PI virus neutralization. We have developed a specific and high-affinity 'tag' that permits the isolation of chemically crosslinked CD4-associated complexes along the pathway of binding and fusion. These complexes will be analyzed biochemically and immunochemically to define the temporal and functional sequence of envelope-CD4-coreceptor interactions. Thirdly, these complexes will be assessed in the transgenic mouse vaccination model to define structures capable of eliciting PI virus neutralizing antibodies. Finally, 'fusion-competent' vaccines and/or purified envelope-CD4-coreceptor complexes will be used in transgenic mice to generate monoclonal antibodies that mediate PI virus neutralization. These fusion-dependent monoclonal antibodies will be used to dissect more finely the molecular pathway of HIV envelope protein binding and fusion. At the conclusion of these studies, we will have developed an integrated description of envelope-CD4-coreceptor function and immunogenicity, towards the definition of critical structures and epitopes for inclusion in novel HIV vaccines capable of eliciting PI virus neutralizing antibodies.